For diagnostic purposes, a commonly used tool is a complete blood count (CBC). The CBC can be divided into three important types of cells that are used for analysis. It is the white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes). By determining important features of erythrocytes, such as mean corpuscular volume (MCV)—the average volume of the erythrocyte, red blood cell distribution width (RDW)—a measure of the variation of the red blood cell population, mean corpuscular hemoglobin (MCH)—the average amount of hemoglobin per erythrocyte, oxygen saturation (SO2)—a measure of the percentage of hemoglobin binding sites in the erythrocyte occupied by oxygen and mean corpuscular hemoglobin concentration (MCHC)—the average concentration of hemoglobin in the erythrocyte, it is possible to diagnose a variety of diseases. For instance is anemia classified as microcytic or macrocytic based on whether the MCV value, is above or below the expected normal range. At present these parameters can be measured by complex and expensive systems, e.g. flow-cytometers and confocal laser scanning microscopy, or systems only capable of only measuring one parameter, e.g. impedance volume analyzer instruments.
In the article “Comparative Study of Human Erythrocytes by Digital Holographic Microscopy, Confocal Microscopy, and Impedance Volume Analyzer” by Rappaz et al. (Journal of the International Society for Advancement of Cytometry, 73A:895-903, 2008), a technique is described that demonstrates the applicability of the digital holography microscopy technique, in combination with a decoupling procedure, for the measurements of refractive indices and volumes of intact individual erythrocytes. Further the decoupling procedure is explained in greater detail in the article “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy” by Rappaz et al. (OPTICS EXPRESS, Vol. 13, No. 23, 936-9373, 2005), where an experimental protocol called “decoupling procedure” is presented with the aim of measuring separately the integral refractive index and the cellular thickness from the quantitative phase images of living cells.
Thus, in order to calculate erythrocyte parameters such as MCV and/or MCHC and/or RDW and/or SO2 and/or MCH it is necessary to have the erythrocyte(s) in a solution and perform an exchange of said solution to a second solution, where the refractive index of the two solutions must be known. This is a laborious and time consuming approach where it requires substantial laboratory skills in performing this without for example contaminating the erythrocytes. Further, the process of exchanging solutions is an additional step that increases the risk of a mishap in the measurement by user interaction. An additional draw back when exchanging solutions, is the fact that all the erythrocytes present in the sample with the first solution will probably not be present after the washing out of the first solution and replacement with the second solution, meaning that there will be a loss, or contamination, of the sample. This reduction of erythrocytes in the solution exchange process, are attributed to that it is necessary to allow the erythrocytes to settle and adhere to the observation vessel before doing the first measurement. If the erythrocytes do not adhere properly, they will unbind and wash out with the first solution and/or move to a new position and adhere on the observation vessel. This will consequently affect the quality of the reconstructed wave front of the erythrocytes by introducing artifacts. Further, the exchange and presence of fluids could affect the erythrocytes morphology and/or desired behavior in an unwanted way. The observation vessel must also only be used one time, so not to contaminate the measurement by for example diluting and/or mixing the solutions and thereby influencing the refractive index to become an unknown instead of as assumed known factor. Further, producing observation vessels with a specific height is very difficult with one time consumables with the required accuracy in the nm range.
Hence, an improved method of determining said parameters is advantageous, and in particular a faster, easier and more reliable method is desired.